When fluid operations are performed in small reaction plates such as microtiter plates, typically one biological reaction is carried out in each well of the plate. Some processes, however, require successive reactions, the reactants or products of one or more of which may be incompatible with subsequent reactions, thereby necessitating the introduction of intervening processes in which such undesired reactants or products are removed or inactivated. For example, genotyping process by the single base extension method requires three biological reactions (i.e., polymerase chain reaction (PCR), enzymatic purification and microsequencing (MIS)). It is efficient and beneficial to perform two or more of these successive reactions in the same well of one unique microtiter plate, a major problem with performing successive reactions in a single well, however, is inter-reaction contamination, i.e., the contamination of subsequent reactions by biomolecules from previous reactions.
For example, one commonly encountered problem in genotyping is contamination by deoxynucleotides (dNTPs) left over from amplification reactions such as PCR of subsequent reactions, such as microsequencing. To address this problem, an enzymatic purification step is typically performed, in which enzymes such as alkaline phosphatase are added to remove dNTPs left over from the PCR. The efficiency of such enzymatic purification reactions is critical, as any dNTPs present in the microsequencing reaction can extend the microsequencing primer beyond the SNP site, thereby allowing the labeled ddNTP to be incorporated downstream of the SNP, leading to errors in genotyping. Similar problems may be encountered in any process involving successive enzymatic reactions. To overcome such problems, the duration of the enzymatic purification step may be extended, the amount of alkaline phosphatase may be increased, or the reaction mixture may be transferred between successive reaction steps, yet in practice none of these potential solutions has been successful in eliminating the problem of contamination.
Clearly, there is a great need in the art for novel methods of decreasing contamination of successive reactions within experimental biological processes. The present invention addresses these and other needs.